Malaria causes an estimated 500 million clinical cases, up to 2.7 million deaths, and a loss of > 1% of GDP in Africa annually, and poses a serious concern for travelers and military. Sanaria's goal is to develop and commercialize a >90% protective attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) vaccine for 2 primary markets with a potential for >$1 billion annual revenues: 1) travelers from the developed world and 2) infants, and young children in the developing world. In limited trials, immunization with radiation-attenuated (RA) PfSPZ has protected >90% of human volunteers against experimental Pf challenge for at least 10 months after the last immunization. No other experimental malaria vaccine has given comparable protection. Heretofore, the development of a RA PfSPZ vaccine has been considered impractical, because it was not considered feasible to: 1) immunize humans by a clinically acceptable parenteral route, 2) produce adequate quantities of PfSPZ, and 3) practically produce and characterize the aseptic, purified, stable, cryopreserved RA PfSPZ needed to meet regulatory and commercial standards for a vaccine. Sanaria has developed methods that have overcome these problems, met with the FDA regarding plans for an IND, and plans to assess the safety, immunogenicity, and protective efficacy of a RA PfSZP vaccine in humans in 2007. Recently it has been demonstrated that it is possible to attenuate P. berghei (Pb) rodent malaria parasites by genetic manipulation. Two groups have independently demonstrated that immunization with parasites deficient in any of 3 distinct Pb genes (PbUIS3, PbUIS4, and Pb36p) protects mice against malaria. Genetically attenuated (GA) PfSPZ have potential advantages over RA PfSPZ from safety and quality control perspectives, and could be a complement to or substitute for the RA PfSPZ vaccine. We have generated a non-clonal population of P. falciparum parasites, of which the PbUIS3 orthologue has been genetically disrupted. This work will now be extended to the other genes listed above and, if necessary, others being currently investigated. The aims of this Phase I SBIR are to, 1) Produce clones of Pf in which the Pf orthologues of PbUIS3, PbUIS4, and Pb36p have been individually knocked out, 2) Demonstrate that at least one of the knock-out parasites infects mosquitoes and produces PfSPZs, and 3) Demonstrate that these PfSPZs invade human hepatocytes in vitro, but are arrested in their development. In subsequent phases it will be established that the parasites do not cause blood stage infection in humans, and when administered in multiple doses as a vaccine, protect humans against challenge with infectious PfSPZs. Once the principle has been proven for one knock-out parasite, other target genes will be assessed alone or in combination in an effort to improve the safety and efficacy of GA PfSPZ as a human vaccine. Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Sanaria's goal is to develop and commercialize a > 90% protective malaria vaccine for primary markets with a potential for > $1 billion annual revenues; 1) Travelers from the developed world, and 2) Infants, young children, and adolescent girls in the developing world. Success in this project has the potential to lead to development of a safe, effective, attenuated malaria vaccine. [unreadable] [unreadable] [unreadable]